基因工程大肠杆菌BL21产β-甘露聚糖酶的代谢流研究
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摘要
该研究基于化学计量平衡建立重组大肠杆菌(Escherichia coli)BL21产β-甘露聚糖酶的代谢流模型,通过比较模型预测的CO2释放通量值和实验测定的CO_2释放通量值验证该模型的可靠性,分析菌体合成通量和β-甘露聚糖酶合成通量分别最大时的极端代谢流分布,并对代谢网络节点(葡萄糖-6-磷酸、丙酮酸)进行分析。结果表明,模型预测的CO_2释放通量值和实验测定的CO_2释放通量值无显著差异(P>0.05),模型可靠;通过极端代谢流分析得出,最大菌体合成通量为9.39 h~(-1),最大β-甘露聚糖酶合成通量为0.18 mmol/(g·h);通过代谢网络节点分析得出,葡萄糖-6-磷酸为柔性节点,丙酮酸为刚性节点,为提高β-甘露聚糖酶的生产能力提供理论依据。
The metabolic flux model of β-mannanase production by recombinant Escherichia coli BL21 was constructed based on stoichiometric balance. The reliability of the model was verified by comparing the predicted CO_2 emission flux value by the model with the determined CO_2 emission flux value. And the extreme metabolic flux distributions were analyzed when the thallus synthesis flux and β-mannanase synthesis flux were the maximal, respectively. Then the metabolic network nodes including glucose-6-phosphate and pyruvate were also studied. The results showed that there was no significant difference between the predicted CO_2 emission flux value by the model and the determined CO_2 emission flux value(P>0.05), and the model was reliable. The analysis results of extreme metabolic flux distributions showed that the maximum thallus synthesis flux and enzyme synthesis flux were 9.39 h~(-1) and 0.18 mmol/(g·h), respectively. The analysis results of the metabolic network nodes showed that the glucose-6-phosphate node was a flexible node and the pyruvate node was a rigid node, which provided a theoretical basis for improving the production capacity ofβ-mannanase.
The metabolic flux model of β-mannanase production by recombinant Escherichia coli BL21 was constructed based on stoichiometric balance. The reliability of the model was verified by comparing the predicted CO_2 emission flux value by the model with the determined CO_2 emission flux value. And the extreme metabolic flux distributions were analyzed when the thallus synthesis flux and β-mannanase synthesis flux were the maximal, respectively. Then the metabolic network nodes including glucose-6-phosphate and pyruvate were also studied. The results showed that there was no significant difference between the predicted CO_2 emission flux value by the model and the determined CO_2 emission flux value(P>0.05), and the model was reliable. The analysis results of extreme metabolic flux distributions showed that the maximum thallus synthesis flux and enzyme synthesis flux were 9.39 h~(-1) and 0.18 mmol/(g·h), respectively. The analysis results of the metabolic network nodes showed that the glucose-6-phosphate node was a flexible node and the pyruvate node was a rigid node, which provided a theoretical basis for improving the production capacity ofβ-mannanase.
引文
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[15]谭松林.反相超高效液相色谱法测定水质中的乙酸含量[J].绿色科技,2017(20):40-41.
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[19]黎明,唐奇,李东霞,等.谷氨酸棒杆菌发酵生产1,5-戊二胺的代谢流分析[J].天津科技大学学报,2015(1):9-13.
[20]QUIRóS M,MART魱NEZ-MORENO R,ALBIOL J,et al.Metabolic flux analysis during the exponential growth phase of Saccharomyces cerevisiae in wine fermentations[J].Plos One,2013,8(8):e71909.
[2]许牡丹,杨伟东,许宝红,等.微生物β-甘露聚糖酶的制备与应用研究进展[J].动物医学进展,2006,27(9):31-34.
[3]杨苗.β-甘露聚糖酶产生菌的筛选鉴定、产酶条件优化及酶学特性研究[D].武汉:湖北工业大学,2016.
[4]唐存多.β-甘露聚糖酶的基因克隆、分子改造及低聚甘露糖的酶法制备[D].无锡:江南大学,2013.
[5]高振,熊强,徐晴,等.代谢通量分析在酶合成过程中的应用研究进展[J].化工进展,2013,32(7):1625-1628.
[6]李智涛,卢志洪,吕扬勇,等.谷氨酸棒杆菌S9114在不同溶氧条件下发酵生产谷氨酸的代谢流分析[J].中国酿造,2010,29(10):72-76.
[7]张洪志,徐庆阳,曹华杰,等.α-淀粉酶抑制剂生物合成途径和代谢流分析[J].食品科学,2017,38(4):118-124.
[8]SARMA S,ANAND A,DUBEY V K,et al.Metabolic flux network analysis of hydrogen production from crude glycerol by Clostridium pasteurianum[J].Bioresource Technol,2017,242:169-177.
[9]WU X,YAO H,LIU Q,et al.Producing acetic acid of acetobacter pasteurianus by fermentation characteristics and metabolic flux analysis[J].Appl Biochem and Biotech,2018,186(1):1-16.
[10]刘朝辉,齐崴,何志敏.地衣芽孢杆菌发酵生产β-甘露聚糖酶的代谢通量分析[J].过程工程学报,2007,7(6):1163-1168.
[11]潘冬瑞,李啸,张瑶,等.重组大肠杆菌E.coli P84A/MC1061发酵生产卤醇脱卤酶的研究[J].天津农业科学,2013,19(7):6-9.
[12]庞伟.13C标记技术测定Escherichia coli TUQ2厌氧中心碳代谢途径通量分布[D].天津:天津大学,2008.
[13]廖祥兵,陈晓明,肖伟,等.DNS法定量测定还原糖的波长选择[J].中国农学通报,2017,33(15):144-149.
[14]谢涛,廖安平,黄春妤,等.浊度法快速测定甘露醇发酵液中的生物量[J].广西民族大学学报:自然科学版,2008,14(3):75-77.
[15]谭松林.反相超高效液相色谱法测定水质中的乙酸含量[J].绿色科技,2017(20):40-41.
[16]张建新,穆广亚,陈琳,等.类芽孢杆菌产β-甘露聚糖酶的分离与纯化[J].中国酿造,2016,35(10):68-71.
[17]?ZKAN P,SARIYAR B,譈TK譈R F魻,et al.Metabolic flux analysis of recombinant protein overproduction in Escherichia coli[J].Biochem Eng J,2005,22(2):167-195.
[18]KLAMT S,KAMP A V.An application programming interface for Cell Net Analyzer[J].Biosystems,2011,105(2):162-168.
[19]黎明,唐奇,李东霞,等.谷氨酸棒杆菌发酵生产1,5-戊二胺的代谢流分析[J].天津科技大学学报,2015(1):9-13.
[20]QUIRóS M,MART魱NEZ-MORENO R,ALBIOL J,et al.Metabolic flux analysis during the exponential growth phase of Saccharomyces cerevisiae in wine fermentations[J].Plos One,2013,8(8):e71909.